High throughput screening of ion channels

ABSTRACT

Multi-well plates having contoured well designs allow multi-stage high throughput parallel assaying of ion channels or ion transporters. A well of a multi-well plate has a bottom region that is sized and shaped to simultaneous accommodate a sensing electrode and a pipette for delivering, e.g., test compounds, wash fluid, and optionally ligands. Such multi-well plates may be coupled with an instrument having a pipette head and an electrode plate. Such arrangement facilitates fluidic contact between cells and fluids provided via a pipette. It also facilitates washing of wells with buffers or other wash solutions to allow serial exposure of test cells to various reagents or other stimuli. Generally, the design allows control and test experiments to be performed on the same cell (or cells) in a single well.

RELATED APPLICATIONS

This application is a divisional application of a co-pending U.S. patentapplication Ser. No. 12/757,499, filed on Apr. 9, 2010, and titled “HighThroughput Screening of Ion Channels,” the content of which isincorporated by reference herein in its entirety.

BACKGROUND

Ion channels are membrane proteins that are present in every cell ofevery living organism. The channels control flow of ions into and out ofthe cells and thus play a crucial role in cell functioning. Notsurprisingly, ion channels constitute a very important class of drugtargets. To efficiently develop new drugs, researchers need methods anddevices that allow high-throughput screening of compounds (drugcandidates) by their action on ion channels. Such drug screening methodsand devices require mechanisms for measuring an ion channel's activityand mechanisms for applying compounds to the ion channels.

Ion channels are commonly studied with a technique called patchclamping. This technique involves measuring electrical signals (currentsand/or voltages) from individual cells. The cells are arranged in anapparatus such that the magnitude of the electrical signal is directlyrelated to the state of the ion channels, and in particular how muchcurrent they allow to pass. Thus, the technique allows direct electricalmeasurement of ion channel events in living cells, cell membranes andartificial membranes.

The whole-cell and perforated patch configurations of the patch-clampare widely accepted as providing the best methods of measuring ionchannel activity for drug screening. In these methods ion currentsflowing through ion channels are measured directly and with highresolution by sensitive current amplifiers.

Unfortunately, current patch clamp instrumentation suffers from avariety of shortcomings, particularly with regard to high-throughputscreening of ion channels, and particularly ligand-gated ion channels.

SUMMARY

Certain embodiments disclosed herein pertain to instruments, multi-wellplates, and associated well designs allowing high throughput parallelassaying of ion channels and ion transporters. Each well of a multi-wellplate has a bottom region that is sized and shaped to simultaneousaccommodate a sensing electrode and a pipette for delivering, e.g., testcompounds, wash fluid, and optionally ligands (for ligand-gated ionchannel assays). Such multi-well plates may be coupled with aninstrument having a pipette head and an electrode plate to providevarious advantages. First, such arrangement allows immediate measurementof current after application of a ligand through the pipette tofacilitate assaying of ligand-gated ion channels. Second, it permitsrelatively complicated assay protocols in which a single well (and itsassociated cell) can be used for both the control and test experimentson ion channels, including ligand-gated ion channels. Additionally, somewell designs contain contoured features that facilitate fluidic contactbetween cells and fluids provided via a pipette. This improves washingof wells and cells with buffers or other wash solutions to allow serialexposure of test cells to various reagents or other stimuli. Generally,the design allows control and test experiments to be performed on thesame cell (or cells) in a single well. This is particularly useful forligand-gated ion channels.

In various embodiments, an assembled device includes one or moreapertures at the bottom of each well, a plenum below the wells, cellswith multiple ion channels sealed to the apertures, a sensing electrodesin the wells beside the cell and a pipette above the cell, and finally,electronics associated with the sensing electrode.

In certain embodiments, the invention pertains to multi-well plateshaving a plurality of wells, in which at least one well of themulti-well plate has a bottom characterized by the following features:(a) a cell cavity sized and shaped to accommodate a pipette tip, and (b)an electrode pocket sized and shaped to accommodate a sensing electrode.In some cases, the cell cavity and the electrode pocket are arranged topermit simultaneous accommodation of the pipette tip and the sensingelectrode. Further, the well bottom may provide a fluidic connectionbetween the electrode pocket and the cell cavity. Typically, the cellcavity includes one or more cell sealing apertures in a bottom surfaceof the cell cavity.

In various embodiments, the cell cavity comprises a pipette guide, whichmay have a shape and size for mating with the pipette tip. For example,the pipette guide may be tapered in the vertical direction. In morespecific cases, the pipette guide's shape and size prevents asignificant fraction of fluid dispensed from the pipette tip, when thepipette is inserted in the guide, from flowing upward and out of thepipette guide. In such designs, and when the well includes fluidicconnection between the cell cavity and the electrode pocket, the fluiddispensed from the pipette tip flows primarily into the cell cavity andthen through the electrode pocket prior to exiting into an upper regionof the well. In embodiments employing an electrode plate (described inmore detail below), the pipette guide may be substantially coaxial witha through hole in the electrode plate.

The dimensions of the electrode pocket may depend on variousapplication-specific features, including the size of the wells, thenumber of wells in the multi-well plate, the size of the electrode, thedesired fluidic coupling between the cell cavity and the electrode, andthe like. In a specific embodiment, the height of the electrode pocket,in the vertical direction, is between about 0.2 and 2 mm. In anotherspecific embodiment, the center-to-center distance between the electrodepocket and the cell cavity is between about 1 and 5 mm.

Similarly, the dimensions of the cell cavity may depend onapplication-specific features including those listed for the electrodepocket as well as certain pipette-specific features such as the size andshape of the pipette tips (e.g., the pipette taper), and the splayassociated with a pipette head. In certain specific embodiments, theheight of the cell cavity, in the vertical direction, is between about0.2 and 2 mm. Further, the diameter or width of the cell cavity may bebetween about 0.5 and 2 mm. In certain specific embodiments, the cellcavity has a size and shape such that when a pipette tip engages withthe cell cavity, the pipette tip comes within about 0.5 mm or less fromthe bottom of the cell cavity.

Another aspect of the invention pertains to a patch clamp apparatus thatmay be characterized by the following features: (a) an electrode platecomprising a plurality of sensing electrodes and a plurality ofassociated through holes sized and positioned to accommodate pipettetips directed from a pipettor head; and (b) a multi-well platecomprising a plurality of wells, each arranged to align with oneelectrode and one through hole of the electrode plate. Further, at leastone well of the multi-well plate includes a cell cavity sized and shapedto accommodate its pipette tip. Typically, each well of the multi-wellplate also includes an aperture for sealing a patch of membrane in thewell. Further, the sensing electrodes of the electrode plate aretypically arranged to provide one electrode per well of an SBS compliantplate having 96, 384, or 1536 wells. Typically, the electrode plateincludes a plurality of contacts for providing electrical connectionbetween the sensing electrodes and associated sensing and recordingelectronics.

In various embodiments, the at least one well of the multi-well plateincludes an electrode pocket sized and shaped to accommodate a sensingelectrode of the electrode plate. As with the first aspect describedabove, the cell cavity and the electrode pocket may be arranged topermit simultaneous accommodation of the pipette tip and the sensingelectrode. Also, as described above, the cell cavity may include apipette guide having a shape and size for mating with a pipette tipdirected into the at least one well. Thus, in certain embodiments, thepipette guide may be substantially coaxial with a corresponding throughhole in the electrode plate.

Another aspect of the invention pertains to methods of conducting apatch clamp assay in which the method is characterized by the followingoperations: (a) providing a cell sealed to an aperture on the bottom ofa well; (b) exposing the cell to a first solution; (c) measuring a firstelectrical signal from the cell while or after the cell is exposed tothe first solution; (d) delivering fresh solution to the bottom of thewell from a pipette inserted in the well; (e) raising the pipette withinthe well, without removing the pipette, and drawing liquid from an upperregion of the well into the pipette; and (f) removing the liquid drawninto the pipette from the well. In various embodiments, the methodsadditionally include exposing the cell to a second solution to afterremoving the liquid in (f) and measuring a second electrical signal fromthe cell while or after the cell is exposed to the second solution. Invarious embodiments, the first electrical signal provides a controlmeasurement and the second electrical signal provides a testmeasurement. In further embodiments, measuring a first electrical signalin (c) is performed immediately upon introduction the first solution tothe well. Additionally, measuring the first electrical signal mayinvolve detecting the current from a sensing electrode in the well.

The above method embodiments may be practiced with a plates andapparatus as described above. For example, wells used in the method mayhave a bottom structure defining a cell cavity as described above. Infurther embodiments, the size and shape of the cell cavity include oneor more of the features described above, including features relating toa pipette guide. The method embodiments may also optionally employ awell having an electrode pocket as described above. The dimensions,fluidic coupling arrangement and other features described above may beavailable when practicing the methods described here.

In yet another aspect, the invention pertains to methods of conductingpatch clamp assays on ligand-gated ion channels. Such methods may becharacterized by the following operations: (a) sealing a patch ofmembrane containing at least one ligand-gated ion channel to an apertureon in an assay well; (b) delivering a ligand to the well and measuringan electrical signal resulting from exposing the ligand-gated ionchannel to the ligand; (c) pipetting fresh solution to the bottom of thewell from a pipette inserted into the well to thereby bathe the patch ofmembrane with the fresh solution; (d) moving the pipette to a differentposition in the well and removing old solution containing the ligandfrom the well; (e) applying a stimulus to the patch of membrane; and (f)delivering the ligand to the well and measuring an electrical signalresulting from exposing the ligand-gated ion channel to the ligand afterapplication of the stimulus. In normal operation in a multi-well plate,the methods will involve repeating the sealing of a patch of membrane in(a) for a plurality of patches in a plurality of wells in a multi-wellplate.

In various embodiments, (b) involves immediately measuring theelectrical signal upon delivering the ligand to the well. Additionally,the ligand-gated ion channel may be allowed to resensitize after (c) and(d) are complete and prior to performing additional operations.Frequently, the stimulus in (a) involves applying a pharmaceuticallyactive compound or biologic material.

The disclosed LGIC assay methods may be practiced with a plates andapparatus as described above. As with the other method embodiments,wells may have a bottom structure defining a cell cavity and/orelectrode pocket as described above. Further, the size and shape(including recited dimensions) of the cell cavity, electrode pocket,and/or pipette guide may be as specified above.

These and other features and advantages will be described in more detailbelow with reference to the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic depictions of a multi-well apparatus forconducting multiple patch clamp experiments in parallel.

FIG. 2 depicts the response profile of a typical ligand-gated ionchannel.

FIG. 3 depicts an instrument including a pipette head and multi-welltest station for performing high throughput patch claim assays.

FIG. 4A presents an exploded view of a multi-well patch clamp regionincluding a plenum, a patch plate, an electrode plate, and a pipettehead.

FIG. 4B is a top view of patch plate 401 in accordance with certainembodiments.

FIGS. 4C through 4E depict an embodiment of an electrode plate suitablefor use with the patch plate depicted in FIG. 4B.

FIG. 5A presents a diagonal cross-section through a single well in apatch plate.

FIG. 5B depicts the well of FIG. 5A but with the pipette raised to anintermediate elevation in the well to draw solution from upper regionsof the well.

FIG. 5C is a top view of a well having a cell cavity and an electrodepocket.

FIG. 6A is a high-level flow chart of a parallel patch clamp process.

FIG. 6B is a flow chart showing implementation of an assay employingvoltage-gated ion channels.

FIG. 6C is a flow chart showing implementation of an assay employingligand-gated ion channels.

FIG. 6D is a flow chart showing implementation of a well washingprocess.

DESCRIPTION OF A PREFERRED EMBODIMENT

Ion channels generally include two parts; the pore (channel) and aswitch that regulates the conductance of the pore. Ion channels arepassive elements in that once opened, ions flow in the direction ofexisting electrochemical gradients. Ion transporters are similar in thatthey are involved in the transport of ions across the cell membrane,however they differ from ion channels in that energy is required fortheir function and they tend to actively pump against establishedelectrochemical gradients. For convenience, the term ion channel will beused herein to refer to both ion transporters and ion channels.

There are two main types of ion channels: voltage-gated ion channels(VGICs) and ligand-gated ion channels (LGICs). VGICs are activated bychanging the electric voltage across the cellular membrane, while LGICsare activated by action of a chemical compound (ligand) on the channelprotein. Studying LGICs requires rapid exchange of extracellularsolution in the vicinity of patched cells.

An example of an instrument in operational configuration for studyingion channels will now be described with reference to its principalcomponents. These components include a plurality of wells (i.e., morethan one well), each with a cell sealed to an aperture, a pipette fordelivering and removing fluid from the well, and an electrode forsensing electrical signals in the well. In more specific embodimentsthat follow, the instrument will be described in terms of a pipettehead, a patch plate which contains a plurality of wells, and a separateelectrode plate which has multiple electrodes arranged to permitindependent sensing in each of the wells of the patch plate. The patchplates and electrode plates of the present invention are not limited tothe specific structures presented in the drawings. Some related featuresof the disclosed embodiments are presented in U.S. Pat. No. 6,488,829and in PCT Publication PCT/US2005/032044, both incorporated herein byreference in their entireties for their descriptions of patch clampinstrumentation.

FIG. 1A shows, in a schematic format, various features of a multi-wellpatch clamp apparatus which permits simultaneous patch clamp experimentson multiple cells. In the depicted apparatus, a well 7 a is depictedalong with partial depictions of adjacent wells 7 b and 7 c. Each wellis bounded by vertical structural members 19 and a bottom horizontalstructural member 20. The bottom horizontal member 20 includes a numberof apertures including an aperture 9 a in well 7 a and an aperture 9 cin well 7 c. During patch clamp experiments, cells are sealed to theindividual apertures which permit ionic coupling to the cell interior.In the depicted example, a cell 1 a is sealed to aperture 9 a and a cell1 c is sealed to aperture 9 c. Below the bottom horizontal member 20there is a plenum 11, which is designed to hold a fluid, such as anintracellular fluid, during patch clamp experiments. The plenum 11 isbounded on the top by the horizontal member 20 and on the bottom by aparallel horizontal member 32 that includes one or more referenceelectrodes (e.g., reference electrode 13) which are electrically coupledto ground 15.

Each of cells 1 a and 1 c includes a plurality of ion channels 17 whichare evaluated for their response to stimuli in the individual wells ofthe apparatus. A typical patch clamp assay involves sensing a signalassociated with current flowing through the various cells (moreparticularly current flowing through ion channels in the cells)positioned in the well and having a membrane patch contacting theplenum.

In certain embodiments, the patch clamp experiment is a perforated patchexperiment. In this variation of the whole-cell patch clamp experiment,the patch of membrane sealed to the cell aperature is perforated or madepermeable to reduce the resistance across the patch of cell membrane inthe aperture. Of course, this permeabilization does not affect theconductivity of ion channels but reduces the resistance associated withthe lipid components of the membrane in the aperture. The electricalpermeabilization of the membrane patch can be induced in many ways. Insome embodiments, it is accomplished by contacting the patch with aperforating agent. This may be accomplished by, for example, providingsuch agent to the solution within the plenum. Examples of suitableperforating agents include certain lipophilic compounds or antibioticssuch as amphotericin-B, nystatin, or gramicidin. Such chemicals work byforming chemical pores in the cell membrane that are permeable tomonovalent ions such as chloride. Since chloride is the current carryingion for the commonly used Ag/AgCl electrode, these compounds produce alow resistance electrical access to the cell interior.

In some embodiments, a relatively high voltage is applied to perforatethe cell membrane and produce a similar result. In such cases,permeabilization is achieved by applying voltage pulses of sufficientstrength and duration that the membrane sealed within the aperturephysically breaks down. This is commonly referred to as “zapping.”

It should be understood that the invention is not limited to perforatedpatch embodiments. Other types of patch clamp assays such as whole-cellconfigurations, which rupture the membrane patch, may also be employed.

In many assays, each stimulus is evaluated for its effect on the abilityof the ion channel 17 to pass ionic current. The current is sensed bysensing electrodes such as a sense electrode 2 a in well 7 a and a senseelectrode 2 b in well 7 b. Such electrodes are typically silver/silverchloride electrodes which provide an electrical connection to sensingcircuitry in response to a reversible exchange of chloride ions in theassay solution. The measurement circuit is completed via the referenceelectrode 13, which may be a second Ag/AgCl electrode.

Typically, though not necessarily, each sense electrode has its ownassociated sensing electronics. In some cases, however, the multipleelectrodes (often in multiple wells) share sensing electronics. Asshown, the sensing electronics may comprise various elements indicatedcollectively by reference number 21. These elements include ahigh-impedance operational amplifier 23 configured to sense the currentflowing in the circuit and a data recording system 25, which is coupledto the amplifier to record and optionally analyze the electrical signalsfrom the well. A high electrical resistance seal between the aperture 9and the cell membrane permits the current recorded by the amplifier tobe dominated by ions flowing through the cell membrane and not by ionsflowing around the aperture directly into the well solution. A voltagecontroller 27 is designed or configured for applying an external voltagebetween the well electrodes 2 and the reference electrode(s) 13, therebyproviding control of the cell's transmembrane voltage potential.

As shown in FIG. 1A, each well includes a separate pipette, e.g., apipette 3 a disposed in well 7 a and a pipette 3 c disposed in well 7 c.At any given time during a patch clamp experiment, the pipettes may holda particular fluid as appropriate for the current stage of the assay. Insome phases, the pipettes are used to deliver fluid to their respectivewells and in other phases of an assay, the pipettes are used to removefluids from their respective wells. In FIG. 1A, the fluids in thepipettes are depicted by the reference numerals 5: 5 a in pipette 3 aand Sc in pipette 3 c.

The bottom of each well contains an internal structural member such as amember 31 shown in well 7 a which is used to at least partially separatea pipette portion of the well from an electrode portion of the well. Asexplained more fully below, such structures may be employed to defineseparate cavities or pockets in different portions of the well. Goodfluidic communication between the pipettes and associated cells andelectrodes in the individual wells permit simultaneous insertion of apipette and an electrode in a given well at any point during an assay.This design also facilitates acquisition of data in studies ofligand-gated ion channels.

FIG. 1B depicts an embodiment similar to that shown in FIG. 1A exceptthat each of the wells contains multiple apertures: for example,apertures 9 a, 9 a′, and 9 a″ in well 7 a. As shown, each of theapertures is positioned to form a seal with a separate cell. Anembodiment employing this design is referred to as a “population patchclamp” or a “parallel patch clamp” (PPC) because it simultaneouslyconsiders the effect of a single stimulus on multiple different cells inparallel. In such embodiments, a single well supports multiple cellswhose contributions to a signal are collectively sensed. Such designscan provide improved data as they average the contributions of multiplecells, any one of which may behave much differently than the norm.Cell-to-cell variability is often significant; individual cells oftenexhibit great variability in their response to a particular stimulus.Further details of a PPC assay design are provided in U.S. patentapplication Ser. No. 11/222,576, filed on Sep. 9, 2005 (see also PCTPublication PCT/US2005/032044), which is incorporated herein byreference in its entirety for its discussion of PPC technology.

It should be understood that the invention is not limited to the studyof ion channels in cell membranes. Rather, the membrane underconsideration may be from, e.g., any one or more of the followingspecies: cells, vesicles, organelles, cell membrane fragments, andsynthetic membranes, any of which include one or more ion channels. Incertain embodiments, the membranous sample may be substantiallyspherically shaped. The portion of the membranous sample outside of theaperture of the partition may be substantially intact.

It should be understood that the concurrent presence of the sensingelectrode and the pipette in a single well provides various options forassay protocols, particularly protocols appropriate for measuring theeffect of drugs on ligand-gated ion channels. FIG. 2 depicts theresponse profile of a typical LGIC. In the figure, ligand concentrationand transient ionic current through the ion channel are plotted alongthe same time axis (the abscissa axis). Ligand-gated ion channelstypically respond to the application of a ligand within a very shortperiod of time, typically less than a second. Very slow actingligand-gated ion channels respond on the order of one to two seconds.After the response reaches a maximum (as measured by a maximum change incurrent flow), the channel becomes desensitized. The period of timebetween the maximum effect of the ligand on the ion channel anddesensitization is typically also very short, on the order of a fewseconds or less. Therefore, in order to measure the change in currentassociated with the opening or closing of a ligand-gated ion channel,the electrode (the sensing electrode) and the source of ligand shouldboth be present in the well at the same time. Note that there are somechannels with slow or no desensitization.

In some prior designs, only one of the sensing electrode and the pipettecould be included in the well at any one time. In these designs, theelectrode and pipette could not contact the solution in a given welltogether, at the same time. To conduct LGIC assays, such systems firstpipette the ligand into the well (while the sensing electrode is not inthe well), and only later, after the pipette is removed, insert theelectrode into the well. Thus, such systems may miss critical earlyinformation about the operation of the ion channel.

In the present invention, the electrode and pipette separately andindependently, but concurrently, contact the solution in the well. Invarious embodiments discussed herein, a well may include both anelectrode and a pipette at the same time, but at different locations.The pipette has multiple functions in a typical LGIC assay, includingdelivering the ligand. In the disclosed designs, the sensing electrodecan continuously monitor current and provide signal for generating acurrent trace while the pipette delivers ligand to the well, whichimmediately impacts the ligand-gated ion channel.

FIG. 3 is a top view of a particular apparatus for performing highthroughput patch clamp assays. As shown, various stations are availablefor providing reagents, cells, wash solutions, etc. to a multi-wellplate. The depicted apparatus includes a pipette head 313 with multiplepipettes 303 installed, which pipette head can move between a testregion 315, where a multi-well patch plate and associated electrodeplate 317 reside, and other stations straddling this test region, whichother regions include a cell reservoir 319, a wash solution reservoir321, a pipette cleaning station 323, a ligand reservoir 325, and a testcompound or drug station 327. Additionally, the apparatus may include abuffer station 329 containing, e.g., fresh extracellular solution fordelivery to the wells. All of these various stations are provided on achassis or table mounted platform 305 over which the pipette head canmove laterally and vertically under the control of appropriateoperational logic, which may be provided by an appropriately programmedcomputer and/or hard coded logic.

Specifically, the pipette head may be a robotic multi-channel pipettorhead, with disposable or non-disposable tips, such as a standardmulti-channel fluidics pipettor (an example of such a pipettor is apipettorr used in the FLIPR® instrument available from MolecularDevices, LLC of Sunnyvale, Calif.). Disposable plastic or glass pipettesare typically used. The fluidics head may be mounted on an X-Y-Z movingactuator. The actuator moves the fluidics head in such a way as to allowpipettes to line-up with openings above the wells and to reach throughthese openings inside the wells of a patch plate mounted in the testregion. Preferably the fluidics head is able to both aspirate fluid fromthe wells and dispense fluids into the wells.

In general, high throughput implementations described herein employrobotics, data processing and control software, liquid handling devices,and detectors. High-throughput screening allows a researcher to quicklyconduct hundreds, thousands, or millions of biochemical, genetic orpharmacological tests on ion channels. Advanced systems permit automatedapplication of reagents and wash solutions in parallel to high-densitymulti-well plates.

FIG. 4A presents an exploded view of a test region (such as test region315 of FIG. 3). The exploded view shows a plenum 423 comprising a plenumreservoir 433, with the patch plate 401 above it, an electrode plate 411above the patch plate, and finally, a pipette head 313 above theelectrode plate 411. Note that plenum 423 includes multiple disk-shapedreference electrodes 421, such as Ag/AgCl electrodes coupled to ground,and mounted to a bottom surface of a plenum compartment 413. The variouscomponents of this test region will now be described individually, andin more detail

FIG. 4B is a top view of patch plate 401 in accordance with certainembodiments. The patch plate in the depicted figure includes 384separate wells 403 arranged in an array. Of course, plates withdifferent numbers of wells can be used, such as patch plates with 1536different wells or 96 different wells. By using a patch plate, with itslarge number of wells, currents from many cells can be measuredconcurrently, simultaneously and independently, which allows rapidcharacterization of compounds or other stimuli applied to the cells.Each cell (or multiple cells) is separated from the rest of the studiedcells by placing that cell (or cells) in a separate well of the patchplate.

The outside dimensions and shape of the patch plate are, in certainembodiments, compatible with the SBS standard (Society for BiomolecularScreening) for multi-well plates to facilitate handling by standardrobotic equipment. As depicted in FIG. 4B, each well 403 includes a cellcavity 405 in the lower left region of the depicted well and anelectrode pocket 407 disposed to the right of the cell cavity. Furtherdetails of suitable designs for wells 405 will be provided in thedescription that follows.

In certain embodiments the patch plate is assembled from two parts. Oneof the parts is injection molded from an inert, biocompatible andelectrically insulating plastic, such as polycarbonate, polystyrene orany other suitable material. This part defines the top and side walls ofthe wells. It may also define the pipette and electrode pockets orcavities as described below. In a specific example, the bottom of eachwell contains one or two relatively large apertures (e.g., about 0.25 to5 mm, or about 1-2 mm, or in a specific case about 1.5 mm in diameter).One of these apertures should be sized and shaped to accommodate apipette tip. If two apertures are employed, the second should be sizedand shaped to accommodate a sensing electrode. In some embodiments, theelectrode diameter is about 0.5 to 2 mm (e.g., about 1 mm).

The second part of the patch plate is a thin film of electricallyinsulating material, such as glass or plastic film, for example apolyimide film such as Kapton(poly(4,4′-oxydiphenylene-pyromellitimide)) film. Other suitablepolymers include polyethylene terephthalate (PET—e.g., Dupont Mylar™),polycarbonate, polypropylene, and polyethylene. The film is bonded tothe bottom of the molded part, covering the aperture. The film containsmany smaller apertures, one or more per each well. In one embodiment,the bottom of each well of the patch plate contains a singlethrough-hole aperture, with at least one dimension of the aperture(typically the diameter of the hole) smaller than the dimension of thecells (e.g., about 1-10 micrometers). Typically the smallest diameter ofthe hole is approximately 2 micrometers. In another embodiment, eachwell of the patch plate contains a plurality of the through-holeapertures (a typical number of apertures in each well is 64). This is aPPC configuration, which was identified in the embodiment of FIG. 1B.

In some cases, the patch plate is hermetically attached or otherwisemounted to the plenum, such that the apertures at the bottom of eachwell connect the well and the plenum compartment. The hermetic seal maybe provided by an elastic gasket and a frame (e.g., a metal frame) thatcompresses the patch plate to the gasket. After mounting the electrodeplate, the plenum is filled with an intracellular buffer solution. Thewells are filled with extracellular buffer solution. As mentioned, theplenum further contains one or more silver/silver chloride (Ag/AgCl)electrodes connected to electrical zero reference (electrical ground).

FIGS. 4C through 4E depict an embodiment of an electrode plate suitablefor use with the patch plate 401 depicted in FIG. 4B. As shown in FIG.4C, the electrode plate 411 includes a substrate 419, such as a printedcircuit board substrate, an array of electrodes 417 having tips ofelectrodes 415 and an associated array of pipette through holes 413. Foreach separate well in the corresponding patch plate there is anassociated electrode 417 and an associated through hole 413 being incontact with an associated tip of electrode 415. Thus, the electrodes417 and through holes 413 are sized and spaced for the patch plate. In aspecific embodiment, there electrodes are spaced to provide oneelectrode for every well of a 384-well configured SBS compliant plate.In other embodiments, the electrodes are spaced to provide one electrodefor every well of a similarly compliant 96-well or 1536-well plate.

The through holes 413 are arranged to permit access of a separatepipette for each well of patch plate 401. Further, in the depictedexample, the through holes 413 are positioned with respect to electrodes417 so that one pipette tip and one electrode 417 are co-located in eachwell.

In some cases, substrate 419 is a printed circuit board (PCB) onto whichthe array of electrodes is attached, e.g., soldered. In a specificembodiment, each electrode is shaped as, for example, a cylinder,approximately 1 mm in diameter, and made from either silver or someother metal (such as steel) plated with silver. Each electrode iscovered by electrically insulating coating (e.g., Teflon™) except forthe most proximal part (bottom) of the electrode, which remains uncoatedby masking during the coating process. The top part of the electrodes issoldered into the substrate, the bottom, uncoated by Teflon part(approximately 1 mm long) is coated with silver chloride. As indicated,the substrate further contains an array of pipette openings 413, eachhole at a specified distance (e.g., about 2 mm) from each electrode.Examples of certain electrode plate embodiments suitable for use withthe invention are further described in U.S. patent application Ser. No.11/222,576 (see also PCT Publication PCT/US2005/032044), previouslyincorporated by reference.

In the embodiment depicted in FIG. 4D, the electrode plate 411 is partof a larger structure having an outside area 451, which may be a frame,beyond the area occupied by the electrode array, and containingelectrical connections 453, such as gold-plated pads for connectingindividual electrodes with monitoring and control circuitry in thechassis. FIG. 4D is a bottom view of the electrode plate and framestructure with through holes 413 and electrodes 417 shown. In somecases, the electrical connections 453 are pins or springs such asspring-loaded pogo-pin electrical connectors. In such cases, wheninstalled in the apparatus, the spring-loaded pogo-pin connectors matewith contacts on the platform adjacent the test region, providingelectrical connection between the amplifiers and/or voltage controlcircuitry (mounted to device's chassis) and the sense electrodes of theelectrode plate. In alternative embodiments, pogo-pin connectors areprovided on the instrument's chassis and mating pads are provided on theelectrode plate. Regardless of the structure of the contacts on theelectrode plate, each contact 453 is connected by a trace wire (notshown) on the substrate to its respective sense electrode.

In the depicted embodiment, the assembly's outer region 451 is anunderlying portion of a frame such as a metal frame, which providesrigidity to the resulting assembly. Additionally, the frame may containfeatures which facilitate clamping the electrode plate to theinstrument. FIG. 4E shows such frame and electrode plate assembly 461(view from above) including a frame 463, which in turn includes handles465 to facilitate manual installation and removal of the electrodeplate.

The electrode plate together with the frame is mounted (e.g., clamped)to the instrument in such a way that the electrodes engage with thepatch plate. For example, when the frame and electrode plate assembly isplaced on a mating surface of the test surface, the electrodes of theplate insert into corresponding wells of the patch plate providingelectrical connection between the fluid in the wells and the electrodes(typically one electrode per well). The array of contacts on the outerregion of the frame underside concurrently line up with and makeselectrical connection with corresponding contacts of the chassis. Thesecorresponding contacts may be provided on mating component (e.g., aregion of the platform or chassis straddling the test region).

As indicated, the instrument additionally contains current-sensingamplifiers, preferably one amplifier per each well of the patch plate.The instrument contacts are connected to the current amplifiers' inputs,such that when the electrode plate is installed on the instrument theelectrodes of the electrode plate connect the fluid in each well withthe amplifier inputs.

FIG. 5A shows a diagonal cross-section through a single well 501 in apatch plate 401. It also shows an electrode plate with an associatedelectrode 417 disposed in well 501. Additionally, it shows a pipette 503extending through a through hole 413 in electrode plate 411 and intowell 501. Well 501 includes an opening or top 513 and a bottom 515.Proximate the well's bottom 515, there is a contoured region including acell cavity 509 and an electrode pocket 511. In the depicted embodiment,the cell cavity 509 extends to very bottom of well 501 while theelectrode pocket 511 does not. The cell cavity 509 and electrode pocket511 are fluidically coupled to one another near their respectivebottoms.

The cell cavity 509 has as its bottom, the bottom sheet or membrane 515of the well. Within the cell cavity 509, and through bottom membrane515, there is an aperture 510 to which a cell under investigation sealsduring assaying. In a parallel patch clamp design, there will bemultiple apertures 510 in the bottom of cell cavity 509.

As shown, the member 31 forms a top of cell cavity 509, which opens at athrough hole 514 into the upper portion of well 501 at a mid-elevationshelf defined by a relatively flat substantially horizontal region 521.Directly below region 521, cell cavity 509 is defined by a pipette guide519 which, in the depicted embodiment, is a generally conical orfunnel-shaped pipette catching feature designed to direct or positionthe pipette tip 505 into a location proximate aperture 510. Bypositioning the tip of an entering pipette at the through hole 514 tothe cell cavity, the pipette guide ensures consistent and direct fluidiccommunication between the pipette tip and cells on aperture 510.Typically, pipette guide 519 is substantially coaxial with thecorresponding opening (pipette through hole) in the electrode platesubstrate.

As mentioned, the instrument may include a robotic pipettor head withmultiple pipettes, such as disposable plastic pipettes. The fluidicshead aligns the pipettes with openings in the electrode plate allowingthe pipettes to reach through these openings and inside the wells of thepatch plate. When a pipette is inserted into the well by the fluidicsrobot, the pipette is captured by a pipette guide, which registers thetip of the pipette. Multichannel fluid pipettors usually exhibit certainpositional errors (splay) of the tips of the pipettes, especially withdisposable plastic pipettes, at least because the pipette tips aredimensionally imprecise. In the absence of the pipette guide this splayresults in variability of the distance between the tip orifice of thepipettes and the cells in the different wells, causing undesirablevariability of fluid exchange. The pipette guide centers the tip of thepipette in the well, reducing this variability.

As indicated, the bottom of well 501 includes two openings to the upperregion of the well, one serving as a pipette guide, and the otherserving as a pocket for the electrode (to the right in FIG. 5A). Thecell cavity and electrode pocket promote fluid exchange at the verybottom of the well, where the patched cell(s) are located. In certainembodiments, the pipette guide 519 forms a tight (or substantiallytight) seal with the pipette tip 505. This provides a clearly definedflow path for fluid delivered from the pipette. Specifically, when thefluid is dispensed into the well from the pipette tip, the fluid flowsthrough the cell cavity 509, thus being directly applied to the cells.The fluid then flows into the electrode pocket 511 and exits into thewell above.

This design facilitates replacement of used or old fluid in wells whentransitioning from one phase of a patch clamp experiment to the next.For example, the design permits the replacement of ligand containingsolution with fresh ligand-free extracellular buffer or wash solutionwhen transitioning between a control phase and a test phase of an assay.Generally, the fluid entering the cell cavity through the pipette guidereplaces the fluid surrounding the cell (or cells). The process ofdelivering fluid from a pipette in the cell cavity flushes the oldfluid, and possibly some excess of the new fluid, which flows throughthe electrode pocket and then into the top part of the well. Thus, thepipette guide, the cell cavity and the electrode pocket create aflow-through channel in the well. Flowing the liquid through thischannel results in efficient and fast fluid exchange in the vicinity ofthe cell or cells. In certain embodiments, the size and shape cellcavity, the electrode pocket and the fluidic connection therebetweendefine a microfluidic flow passage.

In various embodiments, the fluidics head is able to dispense fluidsinto wells and/or aspirate the fluid from the wells of the patch plate(by, e.g., creating a vacuum when the pipettes are raised within thewells 501). As with FIG. 5A, FIG. 5B depicts well 501 together with theelectrode plate electrode 417 and pipette 503. However, in thisdepiction, pipette 503 has been raised to an intermediate elevation inwell 501. Concurrently with or shortly after raising the pipette 503, amechanism is triggered to aspirate or otherwise draw in some fluid fromthe upper regions of well 501, thereby allowing further cleansing of thecell or cells under consideration in the assay. In certain embodiments,the mechanism is a conventional fluid aspiration mechanism such as a setof moving syringe plungers associated with the pipettes. Note that thepipette tip is lifted up to a position where it draws in the liquid fromthe top portion of the well, i.e., where the excess or “dirty” liquidhas accumulated, leaving behind the “clean” liquid in the bottom cellcavity. This lifting/aspiration process, when coupled with priordelivery of wash or new reagent solution to the well, provides aparticularly effective and efficient way of replacing used solution withfresh solution.

FIG. 5C is a top view of well 501 taken from a similar perspective asdepicted for the entire patch plate in FIG. 4B. As shown in FIG. 5C,well 501 includes the cell cavity 509 together with aperture 510 in thelower left region of well 501, and also includes the electrode pocket511 above and to the right of the cell cavity. Also, as indicated, cellcavity 509 and electrode pocket 511 intersect and are in fluidiccommunication. In other words, the electrode pocket and cell cavitytouch or overlap when viewed from above. The center-to-center distancebetween electrode pocket 511 and cell cavity 509 is depicted by a line561. In certain embodiments, this distance is between about 1 and 4 mm,and more specifically between about 1.5 and 3 mm, typically about 2 mm.The cross sectional area of the fluidic connection between the cellcavity and the electrode pocket may be, for example, about 0.1 to 10mm², or more specifically about 1 to 2 mm².

In certain specific embodiments, the well has an open upper region,which is to say that the upper region of well does not contain chambers,baffles or other internal features. The lower regions of the well,however, are contoured to include certain features, typically at least acell cavity. Often the contoured lower region of the well will alsoinclude an electrode pocket and a fluidic connection between theelectrode pocket and cell cavity, which connection is provided proximatethe bottoms of the pocket and the cavity. While the figures presentedherein depict the shape defined by the outer walls of the well (whenviewed from above) to be generally square, this need not be the case. Incertain embodiments, the shape is generally rectangular, circular,elliptical, etc.

Generally, the electrode pocket is sized and shaped to accommodate thesensing electrode with relatively little excess volume beyond thatnecessary to accommodate the electrode. In certain embodiments, thepocket is generally cylindrical in shape, although it may be polygonal,elliptical, oval, etc. The shape will generally match that of thesensing electrode. In various embodiments, the height of the pocket (inthe vertical direction) may be at most about 5 mm, typically betweenabout 1 and 3 mm. In some cases, the principal width or diameter of thepocket may be between about 0.25 and 5 mm, and often between about 1 and3 mm.

As explained, the cell cavity may open into the upper portions of thewell via a pipette guide. The total vertical depth of the cell cavity,including the pipette guide if present, is typically at least about 2mm, and in specific embodiments between about 0.5 and 5 mm. In typicalexamples, the principal diameter or width of the cell cavity will bebetween about 0.5 and 5 mm, and more specifically between about 1 and 2mm. The shape of the cell cavity may be, for example, round, square,rectangular, elliptical, triangular, or other polygonal shape. Incertain embodiments, the cell cavity width or diameter is illustrated byarrow 563 in FIG. 5C. Aside from the pipette guide, the shape anddimensions of the cell cavity typically will not vary significantlyalong its vertical extent.

Regardless of whether a pipette guide is employed, the cell cavity maybe constructed such that when a pipette tip engages with the cavity, thetip comes within about 0.5 mm or less from the aperture(s) at the bottomof the cavity. This facilitates washing of cells with wash or buffersolution from the pipette. In some cases, the vertical distance occupiedby the cell cavity is between about 10 and 50% of the total height ofthe well. Further, the volume occupied by the cell cavity may be betweenabout 0.5 and 5% of the total volume of the well. In specific examples,the total volume of the cell cavity is between about 500 and 2000nanoliters.

The pipette guide, when present, generally has a shape and size formating with the pipette tip; that is the guide is sized and shaped tocircumferentially engage the pipette tip when the pipette is in a finallowered position. Therefore, it will generally have a taper, such as aconical shape or a pyramidal shape. However, in some embodiments, it mayalso have a blunt shape such as an untapered cylindrical or rectangularshape.

Functionally, the pipette guide may, by virtue of its shape and size,prevent a significant fraction of fluid dispensed from the pipette tip,during normal operation, from flowing upward and out of the pipetteguide. When the well also has a fluidic connection between the cellcavity and the electrode pocket, fluid dispensed from the pipette tippositioned in the pipette guide may flow primarily into the cell cavityand then through the electrode pocket prior to exiting into upperregions of the well. In certain embodiments, the pipette guide isbetween about 0.1 and 5 mm long, and in more specific embodiments,between about 1 and 2 mm long. The diameter or width of the pipetteguide at its lower extent may be between about 0.1 and 3 mm in certaindesigns. More specifically, this diameter or width may be between about0.5 and 2 mm.

In accordance with certain embodiments of the invention, various methodsand protocols are provided for performing patch clamp experiments inparallel using a multi-well system. Certain of these protocols employdistinct sequences for ligand-gated ion channels and voltage-gated ionchannels. Some sequences permit performance of both control and testexperiments in a single well on the same cell or cell populationcontained in that well. A few non-limiting sequences will be describedwith reference to FIGS. 6A-6D. At times, these methods will be describedwith reference to a patch plate or other apparatus described above. Itshould be understood, however, that the invention represented by thesemethods is not, unless otherwise noted, limited to the particularapparatus described above.

For context, FIG. 6A presents a general, high-level, description of aprocess that would be implemented using any of the above describeddevices or other devices not specifically presented herein. As depictedin FIG. 6A, the process begins with an operation 603 where the systemintroduces buffer into the various wells of a multi-well plate such asthe patch plate described herein. The buffer may be extracellular bufferfor example. The introduction of buffer may be performed by, forexample, moving a pipette head such as pipette head 313 shown in FIG. 3to a buffer reservoir, drawing buffer into each of the pipettes in thepipette head, moving the buffer head over to a patch plate, lowering thepipette head so that the individual pipettes enter the wells of thepatch plate, and finally, delivering the buffer from the individualpipettes into the various wells of the patch plate. In embodimentsemploying an electrode plate or similar template having pipette throughholes, the step of lowering the pipette head directs the pipettesthrough the through holes and into the wells. Once in the wells, thepipettes may contact a pipette guide such as that depicted in FIG. 5A.

After buffer is applied to the individual wells, the process continueswith an operation 605 where suction is applied to a plenum below theindividual wells in order to clear out any air that may be trapped atthe edges of the apertures in the bottom of the wells of the patchplate. Operation 605 is optional and may be unnecessary in certaindevice designs. Next in the sequence, the plenum below the patch plateis filled with its own buffer solution. See operation 607. Note that theplenum is typically completely filled with buffer so that buffercontacts the bottom surface of the patch plate.

The buffer provided to the plenum may be, for example, an intracellularbuffer, which should be contrasted with the extracellular buffer of thetype that would be typically introduced into the wells in operation 603.Generally, though not necessarily, the extracellular buffer has acomposition chosen to mimic the extracellular environment in which thecells reside in vivo. The intracellular buffer has a composition chosento mimic that found in the cell interior. For example, the extracellularbuffer may contain sodium ions, chloride ions, and calcium ions. Theintracellular buffer may have a similar composition, but with arelatively higher concentration of potassium ions and a relatively lowerconcentration of sodium ions and calcium ions.

After the plenum is completely filled with buffer, the process nextinvolves delivery of cells to the individual wells. This operation isdepicted in an operation 609 and may be performed by, for example,automated delivery of cells from a cell reservoir to the individualwells via a pipette head.

After the cells are delivered to the individual wells of the patchplate, the cells are sealed against the apertures on the bottoms oftheir respective wells. This may be accomplished by establishing apressure differential between the well and the plenum. This operation isdepicted in the flow chart at a block 611. Next, in the sequence, thepatch of cell membrane in the apertures is perforated by an appropriatemechanism. In some embodiments, this is accomplished by “zapping” thecells with a relatively high voltage. In other embodiments, as explainedabove, it is accomplished by introducing a perforatory compound into theplenum. This operation is shown in the flow chart at a block 613. Theperforatory compound introduces some degree of perforation into thepatch of membrane in the aperture. Typical perforatory compounds arelipophilic compounds including certain antibiotics such as amphotericin.When the perforation is concluded, the device is prepared for conductinga patch clamp assay. Details of the various assays that are performedwith the device and its configuration are described in the flow chartsof FIGS. 6B and 6C. In FIG. 6A, these assays are generally representedby a block 615. After the assays are completed, the process mayoptionally involve disposing the pipettes and/or the patch plate, bothof which may be contaminated with particular cells or compounds used inthe assay. See block 617. Alternatively, the pipettes may also be washedand reused, which is sometimes preferable given that pipettes may beexpensive.

As indicated, certain operations may be performed through automatedmovement of a pipette head. Thus, in accordance with certainembodiments, the operations 603, 609, 615, and 617 of the processdepicted in FIG. 6A are performed via automated movement or pipettes. Ina specific embodiment, operation 603 involves pipette head 313 movingbetween station 329 and test region 315. See FIG. 4A. Similarly,operation 609 involves movement between stations 319 and 315, andoperation 617 involves movement between stations 323 and 315. Operation615, which will be described in more detail below, may involve movementbetween the test region 315 and two or more of the buffer station 329,compound station 325, compound station 327, and wash station 321.

FIG. 6B is a flow chart showing implementation of assay 615 from FIG.6A, when assay 615 employs voltage-gated ion channels. As shown, theprocess of FIG. 6B begins with an operation 621 where the instrumentcontrol system applies a first voltage between the sense electrode andreference electrode in order to activate a voltage-gated ion channel.This experiment is performed without applying a test compound or otherstimulus under investigation. It is intended to obtain a control readingfor the operation of the voltage-gated ion channels under consideration.When the instrument applies the first voltage in operation 621, thesystem concurrently measures current to the sense electrode as shown inan operation 623. This current provides a measure of the control readingfor the assay. Next, in an operation 624, the applied voltage isoptionally removed. Thereafter, the test compounds or other stimulusunder investigation are applied to the individual wells of the patchplate. See block 625. This operation may be performed by an automateddelivery using a pipette head. In many assays, different compounds willbe applied to the different wells of a patch plate.

The compounds applied in operation 625 may be permitted to incubate fora period of time in order to ensure that their effect is registered bythe cells in the assay. Regardless of whether such incubation period isprovided, and if it is provided, for how long it is provided, theprocess continues with re-application of a voltage across the electrodesin order to again trigger opening of the voltage-gated ion channels. Seeblock 627. This voltage may be the same as the voltage applied inoperation 621, but it need not be. After the voltage is applied to theelectrodes, the circuitry associated with the device again measures thecurrent to the sense electrodes. See block 629. This time, the currentvalue provides a test value, which can be compared against the controlvalue, for the cells in the individual wells. At this point, operation615 (FIG. 6A) is complete and the process continues with the optionaldisposal of the patch plate and pipettes as described above.

FIG. 6C describes a different implementation of assay 615, animplementation in which ligand-gated ion channels are assayed. Asdepicted in FIG. 6C, the assay begins with an operation 641 whichapplies a first voltage across the electrodes in the device. This stepmay be optional since most ligand-gated ion channels are not activatedby voltage, but rather by ligands. If a voltage is applied, it may be ofa different value than would be applied to activate a voltage-gated ionchannel in a process such as that depicted in FIG. 6B. After optionalstep 641 is completed, the process continues with application of therequired ligand to the individual wells of the patch plate. See block643. This operation may be performed with an automated pipette head asdescribed above. Note that each of the wells in the patch plate will begiven the same ligand because each of them employs cells having the sameligand-gated ion channel. Immediately after the ligand is applied to thewells, the device measures the current to the sense electrodes in orderto provide a control measurement for each well. This is depicted inblock 645 of FIG. 6C. It is important to immediately measure the currentbecause, as indicated in the discussion of FIG. 2, ligand-gated ionchannels quickly become de-sensitized to ligand and “turn off,” even inthe continued presence of ligand. Note that apparatus of the typedescribed above which employs both a pipette tip and an electrode in asingle well permits this immediate measurement of current.

After taking the control measurement in operation 645, the processcontinues with an operation 647 where the individual wells of the patchplate are washed using a buffer or other wash fluid. This process stepmay be accomplished by automated delivery of wash fluid via pipettes. Aspecific example of a wash process is depicted in FIG. 6D. After thewashing is complete, the process may involve waiting a defined period oftime for the cells to re-sensitize so that the next introduction ofligand will again trigger activation of the ion channels. For certainligand-gated ion channels, this requires waiting a few minutes accordingto operation 649. During this wait, or afterwards, the instrumentapplies the test compounds or other stimuli to the individual wells ofthe patch plate. Again, the compounds or other stimuli may be providedby automated delivery from pipettes. See block 651. Typically, differentstimuli are applied to the different wells of the patch plate.Optionally, the cells are incubated with the compounds or other stimulifor a period of time prior to performing test measurements.

When the cells are ready for the test measurements, the processcontinues with an operation 653 where ligand is applied to each of thewells in the patch plate. As before, the ligand may be provided by anautomated delivery from various pipettes. And, as before, after theligand is delivered to many or all wells, the process continues byimmediately measuring the current to the sense electrodes. See operation655. This current measurement provides test data for comparison againstthe control data obtained in operation 645. After the test data isobtained and appropriately processed, if necessary, operation 615 fromflow chart 6A is completed and the overall process continues asdescribed above.

As should be clear, drug screening, particularly screening for effect onLGICs, often requires repetitive cycles of compound addition andwashout. In accordance with certain embodiments, the washout isaccomplished as depicted in FIG. 6D. Initially, a pipette is filled withbuffer solution (operation 661) and then brought into the well andpositioned in the cell cavity. See operation 663. The solution is thendispensed into the bottom cavity, replacing the old fluid in the cavitywith the wash fluid (operation 665). Excess solution overflows into thewell above the electrode pocket. The pipette may dispense all or most ofthe buffer it carried into the well. After the pipette empties itscontents the fluidics head is lifted up a few millimeters (e.g., about2-10 mm) so that the tip of the pipette exits the pipette guide and ispositioned in the well above the guide (see FIG. 5B) and below the upperlevel of the fluid within the well. See operation 667. At this point,the action of the fluidic head is reversed and the pipette aspirates theliquid from the top part of the well, leaving behind the clean buffer inthe bottom cavity containing the patched cell(s). In other words, theposition of the pipette allows it to draw in liquid from an upper regionof the well into the pipette. Typically, the pipette tip is raised fromthe cell cavity within about 0 to 10 seconds after dispensing the bufferor other solution. Note that in some cases, there is no need to wait atall.

The aspirated liquid is a mixture of the old liquid and the wash liquid.This liquid from the pipettes is then dumped into waste, the pipettesare optionally washed/replaced, and the cycles of washout (dispensingwash liquid into the cell cavity through the pipette guide withsubsequent lifting of the pipette and aspiration of the mixed liquidfrom the well above the pipette guide) are repeated sufficient number oftimes to achieve the required degree of washing.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention be defined by the claims appended hereto and theirequivalents.

1. A multi-well plate comprising a plurality of wells, wherein at leastone well of the multi-well plate comprises a bottom comprising: (a) acell cavity sized and shaped to accommodate a pipette tip, andcomprising a pipette guide shaped and sized for mating with the pipettetip; (b) an electrode pocket sized and shaped to accommodate a sensingelectrode; and (c) a fluidic connection between the cell cavity andelectrode pocket; wherein the cell cavity and the electrode pocket arearranged to permit simultaneous accommodation of the pipette tip and thesensing electrode respectively.
 2. The multi-well plate of claim 1,wherein the cell cavity comprises at least one cell sealing aperture ina bottom surface of the cell cavity. 3-6. (canceled)
 7. The multi-wellplate of claim 6, wherein the pipette guide shape and size prevent asignificant fraction of fluid dispensed from the pipette tip fromflowing upward and out of the pipette guide.
 8. The multi-well plate ofclaim 7, the fluidic connection between the cell cavity and theelectrode pocket allows for fluid dispensed from the pipette tip to flowprimarily into the cell cavity and then through the electrode pocketprior to exiting into an upper region of the well.
 9. The multi-wellplate of claim 1, wherein the pipette guide is substantially coaxialwith a through hole in an electrode plate.
 10. The multi-well plate ofclaim 1, wherein the pipette guide is tapered in the vertical direction.11. The multi-well plate of claim 1, wherein the height of the electrodepocket, in the vertical direction, is between about 0.2 and 2 mm. 12.The multi-well plate of claim 1, wherein the height of the cell cavity,in the vertical direction, is between about 0.2 and 2 mm.
 13. Themulti-well plate of claim 1, wherein the diameter or width of the cellcavity is between about 0.5 and 2 mm.
 14. The multi-well plate of claim1, wherein a center-to-center distance between the electrode pocket andthe cell cavity is between about 1 and 5 mm.
 15. The multi-well plate ofclaim 1, wherein the cell cavity has a size and shape such that when apipette tip engages with the cell cavity, said pipette tip comes withinabout 0.5 mm or less from the bottom of the cell cavity. 16.-39.(canceled)
 40. A method of conducting a patch clamp assay on aligand-gated ion channel, the method comprising: (a) sealing a patch ofmembrane containing at least one ligand-gated ion channel to an apertureon in an assay well which comprises a cell cavity and electrode pocketsized and shaped to accommodate a pipette tip and a sensing electroderespectively and to permit their simultaneous placement within the cellcavity and the electrode pocket; (b) delivering a ligand to the well andmeasuring an electrical signal resulting from exposing the ligand-gatedion channel to the ligand; (c) pipetting fresh solution to a bottom ofthe well from a pipette inserted into the well via a pipette guide toproviding a direct fluidic communication between the pipette and thecell cavity via an opening in the cell cavity and to thereby bathe thepatch of membrane with the fresh solution; (d) moving the pipette withinthe well and removing old solution containing said ligand from the well;(e) applying a stimulus to the patch of membrane; and (f) delivering theligand to the well and measuring an electrical signal resulting fromexposing the ligand-gated ion channel to the ligand after application ofthe stimulus.
 41. The method of claim 40, further comprising repeatingthe sealing of a patch of membrane in (a) for a plurality of patches ina plurality of wells in a multi-well plate.
 42. The method of claim 40,further comprising allowing the ligand-gated ion channel to resensitizeafter (c) and (d) are complete.
 43. The method of claim 40, whereinapplying the stimulus in (a) comprises applying a pharmaceuticallyactive compound or biologic material.
 44. The method of claim 40,wherein (b) comprises immediately measuring the electrical signal upondelivering the ligand to the well.
 45. The method of claim 40, whereinthe assay well comprises an open top sized and shaped to simultaneouslyaccommodate a pipette tip and a sensing electrode.
 46. The method ofclaim 40, wherein the assay well comprises a bottom having a cell cavitysized and shaped to accommodate a pipette tip.
 47. The method of claim46, wherein the assay well further comprises a fluidic connectionbetween the cell cavity and the electrode pocket, whereby, during (c)the fresh solution is dispensed from the pipette tip and flows primarilyinto the cell cavity and then through the electrode pocket prior toexiting into an upper region of the well.
 48. The method of claim 46,wherein the cell cavity comprises the pipette guide having a shape andsize for mating with the pipette tip directed into the at least onewell.
 49. The method of claim 48, wherein the pipette guide issubstantially coaxial with a corresponding through hole in an electrodeplate disposed above the well.
 50. The method of claim 48, wherein thepipette guide is tapered in the vertical direction.
 51. The method ofclaim 46, wherein a height of the cell cavity, in the verticaldirection, is between about 0.2 and 2 mm.